Stress and glucocorticoid hormones, which are released into the circulation following stressful experiences, have been shown to contribute significantly to the manifestation of various psychiatric illnesses including Post- Traumatic Stress Disorder (PTSD) and depression. The brain-derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays a critical role in stress-mediated changes in structural as well as functional plasticity in prefrontal cortex. Deregulation of TrkB expression and signaling in prefrontal cortex, and elevated glucocorticoid levels are linked to schizophrenia, PTSD and depression. In addition, studies have indicated that the chronic treatment effect of glucocorticoids on BDNF signaling is through non-genomic actions as compared to their acute genomic functions. However, the molecular mechanisms that regulate TrkB receptor internalization and turnover remain unknown. The overall goal of this proposal is to determine the role of ubiquitination in glucocorticoid-induced downregulation of the TrkB. We provide initial evidence demonstrating that chronic glucocorticoid induces ubiquitin-proteasome-mediated degradation of TrkB and up-regulation of the ubiquitin E3 ligase c-Cbl. We also provide evidence that the c-Cbl mRNA levels are higher in the dorsolateral prefrontal cortex of schizophrenia and bipolar subjects. We found an increased TrkB ubiquitination and association with c-Cbl in the prefrontal cortex of schizophrenia subjects. We hypothesize that chronic glucocorticoid induces c-Cbl dependent downregulation of TrkB in prefrontal cortex. We will also investigate the role of Sprouty2 in glucocorticoid -induced downregulation of TrkB. Sprouty2 is known to regulate growth factor signaling through its interaction with c-Cbl. Our recent study has found a significant reduction in Sprouty2 mRNA levels in the dorsolateral prefrontal cortex of schizophrenia, bipolar and suicide subjects. We now provide initial evidence that chronic glucocorticoid exposure downregulates Sprouty2 expression. We propose a comprehensive series of studies aimed at examining the role of c-Cbl and Sprouty2 in chronic glucocorticoid -induced TrkB downregulation in primary cortical neurons using co- immunoprecipitation studies, small interfering RNA (siRNA) analysis and immunofluorescence microscopy experiments. Our objective is to define the cellular and molecular mechanisms that underlie the TrkB downregulation following chronic glucocorticoid exposure. Two Specific Aims are proposed. The Specific Aim 1 will test the hypothesis that chronic glucocorticoid-induced TrkB downregulation is mediated through c-Cbl. The Specific Aim 2 will test the hypothesis that overexpression of Sprouty2 attenuates glucocorticoid-induced downregulation of TrkB expression through its interaction with c-Cbl. A better characterization of the role of c-Cbl and Sprouty2 proteins may open up insights into the mechanisms governing TrkB receptor regulation. In addition, such a systematic study will also provide fundamental mechanistic information that is critical to the eventual development of effective therapy for several stress related pathologies, including PTSD, schizophrenia and depression. PUBLIC HEALTH RELEVANCE: This application will explore the mechanism of TrkB receptor regulation under chronic stress. Given the important role of TrkB signaling in brain function, the findings of this exploratory study (R21) may provide avenues to develop newer therapeutic agents for such debilitating disorders PTSD, addiction, autism, depression and schizophrenia.